WO2016117567A1

WO2016117567A1 - Separation material

Info

Publication number: WO2016117567A1
Application number: PCT/JP2016/051460
Authority: WO
Inventors: 優渡邊; 後藤　泰史; 道男佛願; 中西　良一
Original assignee: 日立化成株式会社
Priority date: 2015-01-19
Filing date: 2016-01-19
Publication date: 2016-07-28
Also published as: EP3248677B1; US10898877B2; JP6778381B2; US20180264435A1; EP3248677A4; EP3248677A1; JPWO2016117567A1

Abstract

The present invention provides a separation material that is provided with: porous polymer particles that include styrene and/or divinylbenzene as a monomer unit to the amount of 90 mass% or more in terms of the total monomer quantity; and a coating layer that coats at least a portion of the surface of the porous polymer particles and includes a polymer having a hydroxyl group. Therein, the coating amount of the coating layer is 1-15 mg/m² per unit specific surface area of the porous polymer particles.

Description

Separation material

The present invention relates to a separating material.

Conventionally, when separating and purifying biopolymers typified by proteins, there are generally ion exchangers based on porous synthetic polymers, particles based on cross-linked gels of hydrophilic natural polymers, etc. It is used. The ion exchanger based on the above porous synthetic polymer has an advantage that the pressure resistance during liquid passage is good. However, when this ion exchanger is used for protein separation, nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, resulting in peak asymmetry, or ion exchange by hydrophobic interaction. There is a problem that the protein adsorbed on the body may not be recovered while adsorbed.

On the other hand, in the case of an ion exchanger based on a crosslinked gel of the above-mentioned hydrophilic natural polymer represented by polysaccharides such as dextran and agarose, there is an advantage that there is almost no nonspecific adsorption of protein. However, this ion exchanger swells remarkably in an aqueous solution, and has the disadvantage that the volume change due to the ionic strength of the solution, the volume change between the free acid form and the loaded form is large, and the mechanical strength is not sufficient. . In particular, when a cross-linked gel is used in chromatography, there is a disadvantage that the pressure loss during liquid passage is large and the gel is consolidated by liquid passage.

In order to overcome the drawbacks of having a hydrophilic natural polymer cross-linked gel, attempts have been made so far to combine rigid substances that become skeletons.
For example, a complex in which a gel such as a natural polymer gel is held in the pores of a porous polymer is known in the field of peptide synthesis (see Patent Document 1). Patent Document 1 describes that by using such a complex, the load factor of the reactive substance is increased, and high yield synthesis is possible.

An ion exchanger of a hybrid copolymer in which pores of a so-called macro network structure copolymer are filled with a crosslinked copolymer gel synthesized from a monomer is known (see Patent Document 2). Crosslinked copolymer gels have problems such as pressure loss and volume change when the degree of crosslinking is low. However, by using a hybrid copolymer, liquid flow characteristics are improved, pressure loss is reduced, and ion exchange capacity. Patent Document 2 describes that the leakage behavior is improved.

There has been proposed a composite filler in which a hydrophilic natural polymer cross-linked gel having giant meshes is filled in the pores of an organic synthetic polymer substrate (see Patent Documents 3 and 4).

A technique for synthesizing porous particles composed of glycidyl methacrylate and an acrylic crosslinked monomer is known (see Patent Document 5).

U.S. Pat. No. 4,965,289 U.S. Pat. No. 3,966,489 JP-A-1-254247 US Pat. No. 5,114,577 JP 2009-244067 A

With conventional column packing materials, it is possible to solve all of the problems of natural polymer, such as liquid permeability, polymer particles, such as nonspecific adsorption of proteins such as enzymes, improvement of adsorption amount, and suppression of denaturation. Have difficulty.

Therefore, an object of the present invention is to provide a separation material that ensures liquid permeability and can reduce non-specific adsorption of proteins, increase the amount of adsorption, and suppress denaturation.

The present invention provides the separating material described in [1] to [6] below.
[1] The polymer unit has at least one of styrene and divinylbenzene at 90% by mass or more based on the total amount of the monomer, and has a hydroxyl group that covers at least a part of the surface of the porous polymer particle. And a coating layer containing a polymer, wherein the coating amount by the coating layer is 1 to 15 mg / m ² per unit specific surface area of the porous polymer particles.
[2] The separation material according to [1], wherein the oxygen element ratio on the surface of the separation material is 10 to 50%.
[3] The separation material according to [1] or [2], wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
[4] The separation material according to [3], wherein the polysaccharide is at least one selected from agarose, chitosan, alginic acid, and dextran.
[5] The separating material according to any one of [1] to [4], wherein a polymer having a hydroxyl group is crosslinked.
[6] The separation material according to any one of [1] to [5], wherein the specific surface area of the porous polymer particles is 10 m ² / g or more.
[7] The separation material according to any one of [1] to [6], wherein the mode diameter in the pore size distribution of the porous polymer particles is 0.05 to 1 μm.
[8] A separation column comprising a column and the separation material according to any one of [1] to [7] packed in the column.

According to the present invention, it is possible to provide a separation material that ensures liquid permeability and can reduce non-specific adsorption of proteins, increase the amount of adsorption, and suppress denaturation.

It is sectional drawing which shows one Embodiment of the column for separation.

Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments.

<Separation material>
The separating material of the present embodiment includes porous polymer particles and a coating layer that covers at least part of the surface of the porous polymer particles. In the present specification, the “surface of the porous polymer particle” includes not only the outer surface of the porous polymer particle but also the surface of the pores inside the porous polymer particle.

(Porous polymer particles)
The porous polymer particles of the present embodiment are particles obtained by curing a monomer containing a porosifying agent, and can be synthesized by, for example, conventional suspension polymerization or emulsion polymerization. As the monomer unit, at least one of styrene and divinylbenzene contains 90% by mass or more based on the total monomer amount, and preferably 90% by mass or more of divinylbenzene based on the total monomer amount. By containing a predetermined amount of styrene or divinylbenzene, the pressure resistance tends to be excellent.
The monomer may further contain the following polyfunctional monomers other than styrene and divinylbenzene, monofunctional monomers, and the like.

Examples of polyfunctional monomers other than divinylbenzene include divinyl compounds such as divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination of two or more.

Monofunctional monomers other than styrene include, for example, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4 -Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn- Examples thereof include styrene derivatives such as dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorostyrene. These monofunctional monomers can be used singly or in combination of two or more. Moreover, the styrene derivative which has functional groups, such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group, can also be used.

Examples of the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, alcohols, and the like, which are organic solvents that promote phase separation at the time of polymerization and promote pore formation of particles. It is done. Specific examples include toluene, xylene, diethylbenzene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol and the like. These porous agents can be used singly or in combination of two or more.

The above porosifying agent can be used in an amount of 0 to 200% by mass based on the total mass of the monomer. The porosity of the porous polymer particles can be controlled by the amount of the porous agent. Furthermore, the size and shape of the pores of the porous polymer particles can be controlled by the kind of the porous agent.

Water used as a solvent can be used as a porous agent. When water is used as a porosifying agent, it is possible to make it porous by dissolving an oil-soluble surfactant in the monomer and absorbing water.

Examples of the oil-soluble surfactant used for the porosification include sorbitan monoesters of branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids or chain saturated C12 to C14 fatty acids, such as sorbitan monolaurate, sorbitan Sorbitan monoesters derived from monooleate, sorbitan monomyristate or coconut fatty acid; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, for example di- Glycerol monooleate (for example, diglycerol monoester of C18: 1 (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate or diglycerol monoester of coconut fatty acid Ester; Branch C16 ~ 24 alcohol (e.g., Guerbet alcohols), linear unsaturated C16 ~ C22 alcohols or linear saturated C12 ~ C14 alcohols (e.g., coconut fatty alcohols) diglycerol mono-fatty ethers; and mixtures thereof.

Of these, sorbitan monolaurate (e.g., SPAN 20), preferably greater than about 40% purity, more preferably greater than about 50% purity, and most preferably greater than about 70% purity. Sorbitan monooleate (eg, SPAN 80, preferably about 40% purity, more preferably about 50% purity, most preferably greater than about 70% purity sorbitan monooleate); Glycerol monooleate (eg, greater than about 40% purity, more preferably greater than about 50% purity, most preferably greater than about 70% purity); diglycerol monoisostearate (eg, preferably Is greater than about 40% purity, more preferably greater than about 50% purity Most preferably a diglycerol monoisostearate of greater than about 70% purity; diglycerol monomyristate (preferably greater than about 40% purity, more preferably greater than about 50% purity, most preferably about 70% purity). More preferred sorbitan monomyristate); cocoyl (eg, lauryl, myristoyl, etc.) ethers of diglycerol; and mixtures thereof.

These oil-soluble surfactants are preferably used in the range of 5 to 80% by mass relative to the total mass of the monomer. If the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplet is sufficient, and it is difficult to form a large single hole. Further, when the content of the oil-soluble surfactant is 80% by mass or less, it becomes easier for the porous polymer particles to retain the shape after polymerization.

Examples of the aqueous medium used for the polymerization reaction include water, a mixed medium of water and a water-soluble solvent (for example, lower alcohol), and the like. The aqueous medium may contain a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants can be used.

Examples of the anionic surfactant include fatty acid oils such as sodium oleate and castor oil potassium, alkyl sulfate salts such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfone. Acid salts, alkane sulfonates, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, alkenyl succinates (dipotassium salts), alkyl phosphate esters, naphthalene sulfonate formalin condensates, polyoxyethylene alkylphenyl ether sulfates Salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, polyoxyethylene alkyl sulfates, etc.

Examples of the cationic surfactant include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.

Nonionic surfactants include, for example, hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, or amides. Agents, polyether-modified silicon-based nonionic surfactants such as polyethylene oxide adducts of silicon and polypropylene oxide adducts, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.

Examples of zwitterionic surfactants include hydrocarbon surfactants such as lauryl dimethylamine oxide, phosphate ester surfactants, and phosphite ester surfactants.

Surfactant may be used alone or in combination of two or more. Among the surfactants, anionic surfactants are preferable from the viewpoint of dispersion stability during monomer polymerization.

Examples of the polymerization initiator added as necessary include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butyl peroxide. Organic peroxides such as oxy-2-ethylhexanoate and di-tert-butyl peroxide; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2 ′ -Azo compounds such as azobis (2,4-dimethylvaleronitrile). The polymerization initiator can be used in the range of 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monomer.

The polymerization temperature can be appropriately selected according to the type of monomer and polymerization initiator. The polymerization temperature is preferably 25 to 110 ° C, more preferably 50 to 100 ° C.

In the synthesis of porous polymer particles, a polymer dispersion stabilizer may be used in order to improve the dispersion stability of the particles.

Examples of the polymer dispersion stabilizer include inorganic water-soluble high polymers such as polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), polyvinyl pyrrolidone, tricalcium phosphate (TCP), sodium tripolyphosphate, and the like. Examples include molecular compounds. These can be used individually or in combination of multiple types. Of these, tricalcium phosphate (TCP), polyvinyl alcohol, or polyvinylpyrrolidone is preferred. The addition amount of the polymer dispersion stabilizer is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monomer.

In order to suppress the polymerization of the monomer alone, a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin Bs, citric acid, polyphenols and the like may be used.

The average particle diameter of the porous polymer particles is preferably 300 μm or less, more preferably 150 μm or less, and even more preferably 100 μm or less. Further, the average particle diameter of the porous polymer particles is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more, from the viewpoint of improving liquid permeability.

The coefficient of variation (CV) of the particle size of the porous polymer particles is preferably 3 to 15%, more preferably 5 to 15%, from the viewpoint of improving liquid permeability. More preferably, it is 10%. C. V. As a method for reducing the above, monodispersion by an emulsification apparatus such as a microprocess server (Hitachi Ltd.) can be mentioned.

C. of average particle size and particle size of porous polymer particles or separator V. (Coefficient of variation) can be determined by the following measurement method.
1) Disperse the particles in water (including a dispersant such as a surfactant) using an ultrasonic dispersion device to prepare a dispersion liquid containing 1% by mass of porous polymer particles.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), an average particle size and particle size of C.I. V. (Coefficient of variation) is measured.

The pore volume (porosity) of the porous polymer particles is preferably 30% by volume or more and 70% by volume or less, and 40% by volume or more and 70% by volume based on the total volume (including the pore volume) of the porous polymer particles. % Or less is more preferable. The porous polymer particles preferably have pores having a mode diameter (mode value of pore diameter distribution, maximum frequency pore diameter, average pore diameter) of 0.05 to 1 μm in the pore diameter distribution. The mode diameter in the pore size distribution of the porous polymer particles is more preferably 0.2 μm or more and less than 0.5 μm. When the mode diameter in the pore size distribution is 0.05 μm or more, a substance tends to easily enter the pores. When the mode diameter in the pore diameter distribution is 1 μm or less, the specific surface area is sufficient. These can be adjusted by the above-mentioned porous agent.

The specific surface area of the porous polymer particles is preferably 10 m ² / g or more, and more preferably 30 m ² / g or more. From the viewpoint of higher practicality, the specific surface area is more preferably 35 m ² / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 10 m ² / g or more, the adsorption amount of the substance to be separated tends to increase. The upper limit of the specific surface area of the porous polymer particles is not particularly limited, but can be, for example, 200 m ² / g or less, 100 m ² / g or less.

(Coating layer)
The coating layer of this embodiment contains a polymer having a predetermined amount (coating amount) of hydroxyl groups. By covering the porous polymer particles with a polymer having a predetermined amount of hydroxyl groups, the increase in column pressure can be suppressed, and nonspecific adsorption of proteins can be suppressed, and the protein adsorption of the separation material The amount tends to be good. Furthermore, when the polymer having a hydroxyl group is crosslinked, it is possible to further suppress an increase in column pressure.

In the separation material of this embodiment, the coating amount by the coating layer is 1 to 15 mg / m ² per unit specific surface area of the porous polymer particles, preferably 5 to 15 mg / m ² , and preferably 8 to 15 mg / m 2. ² is more preferable. By setting the coating amount by the coating layer within a predetermined range, it is possible to suppress non-specific adsorption and protein denaturation and improve the adsorption amount when used as a protein separation material. The coating amount by the coating layer can be measured by the method described in the examples.

In the separation material of the present embodiment, the oxygen element ratio on the surface is preferably 10 to 50%, more preferably 15 to 50%, further preferably 20 to 50%, and more preferably 30 to 50%. And particularly preferred. By setting the oxygen element ratio on the surface of the separation material within a predetermined range, the effect of suppressing nonspecific adsorption and protein denaturation and the amount of adsorption when used as a protein separation material are further improved. The oxygen element ratio on the surface of the separating material can be measured by the method described in the examples. The oxygen element ratio on the surface of the separating material is measured by X-ray photoelectron spectroscopy in which the coating layer is irradiated with X-rays from the outside of the separating material.

(Polymer having a hydroxyl group)
The polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and more preferably a hydrophilic polymer. Examples of the polymer having a hydroxyl group include polysaccharides and polyvinyl alcohol. Preferred examples of the polysaccharide include agarose, dextran, cellulose, chitosan, and alginic acid. As the polymer having a hydroxyl group, for example, a polymer having a weight average molecular weight of about 10,000 to 200,000 can be used.

The polymer having a hydroxyl group is preferably a modified product modified with a hydrophobic group from the viewpoint of improving the interfacial adsorption ability. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, and a propyl group. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. A hydrophobic group is introduced by reacting a functional group that reacts with a hydroxyl group (for example, an epoxy group) and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with a polymer having a hydroxyl group by a conventionally known method. Can do.

(Formation method of coating layer)
The coating layer containing a polymer having a hydroxyl group can be formed by, for example, the following method.
First, a polymer solution having a hydroxyl group is adsorbed on the surface of the porous polymer particles. The solvent for the polymer solution having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, but water is the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).
This solution is impregnated into porous polymer particles. In the impregnation method, porous polymer particles are added to a polymer solution having a hydroxyl group and left for a predetermined time. Although the impregnation time varies depending on the surface state of the porous body, the polymer concentration is in an equilibrium state with the external concentration inside the porous body if it is usually impregnated day and night. Then, it wash | cleans with solvents, such as water and alcohol, and the polymer | macromolecule which has the hydroxyl group which is not adsorbed is removed.

(Crosslinking treatment)
Next, a crosslinking agent is added to cause the polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to undergo a crosslinking reaction to form a crosslinked body. That is, the polymer having a hydroxyl group of the separating material may be cross-linked. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.

As a crosslinking agent, for example, an epihalohydrin such as epichlorohydrin, a dialdehyde compound such as glutaraldehyde, a diisocyanate compound such as methylene diisocyanate, a glycidyl compound such as ethylene glycol diglycidyl ether, and two or more functional groups active on a hydroxyl group. The compound which has is mentioned. When a compound having an amino group such as chitosan is used as the polymer having a hydroxyl group, a dihalide such as dichlorooctane can also be used as a crosslinking agent.

A catalyst is usually used for this crosslinking reaction. As the catalyst, a conventionally known catalyst can be appropriately used according to the type of the crosslinking agent. For example, when the crosslinking agent is epichlorohydrin or the like, an alkali such as sodium hydroxide is effective, and in the case of a dialdehyde compound. Mineral acids such as hydrochloric acid are effective.

The crosslinking reaction with the crosslinking agent is usually performed by adding the crosslinking agent to a system in which the separating material is dispersed and suspended in an appropriate medium. When the polysaccharide is used as the polymer having a hydroxyl group, the addition amount of the crosslinking agent is, for example, within a range of 0.1 to 100 moles per unit of one unit of monosaccharide. It can be selected according to performance. Generally, when the addition amount of the crosslinking agent is reduced, the coating layer tends to be easily peeled off from the porous polymer particles. Moreover, when the addition amount of a crosslinking agent is excessive and the reaction rate with the polymer which has a hydroxyl group is high, the characteristic of the polymer which has a hydroxyl group of a raw material tends to be impaired.

The amount of the catalyst used varies depending on the type of the crosslinking agent. Usually, when a polysaccharide is used as the polymer having a hydroxyl group, if one unit of the monosaccharide forming the polysaccharide is 1 mole, It is preferably used in the range of 0.01 to 10 mole times, more preferably 0.1 to 5 mole times.

For example, when the cross-linking reaction condition is a temperature condition, the temperature of the reaction system is raised, and the cross-linking reaction occurs when the temperature reaches the reaction temperature.

As a medium for dispersing and suspending porous polymer particles impregnated with a polymer solution having a hydroxyl group, the polymer or crosslinking agent is not extracted from the impregnated polymer solution, and a crosslinking reaction is performed. Must be inert. Specific examples thereof include water, alcohol, toluene, dichlorobenzene, nitromethane and the like.

The crosslinking reaction is usually performed at a temperature in the range of 5 to 90 ° C. for 1 to 10 hours. The temperature is preferably in the range of 30 to 90 ° C.

After completion of the crosslinking reaction, the produced particles are filtered, washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove unreacted polymer, suspending medium, etc. A separation material in which at least a part of the surface is coated with a coating layer containing a polymer having a hydroxyl group is obtained.

(Introduction of ion exchange groups)
The separation material having a coating layer can be used for ion exchange purification, affinity purification, etc. by introducing ion exchange groups, ligands (protein A), etc. via hydroxyl groups on the surface. Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.

Examples of the halogenated alkyl compound include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid and sodium salts thereof, primary, secondary or tertiary amines having at least one halogenated alkyl group such as diethylaminoethyl chloride, halogen And quaternary ammonium hydrochloride having an alkyl group. These halogenated alkyl compounds are preferably bromides or chlorides. The amount of the halogenated alkyl compound used is preferably 0.2% by mass or more based on the total mass of the separating material imparting ion exchange groups.

For the introduction of ion exchange groups, it is effective to use an organic solvent in order to promote the reaction. Examples of the organic solvent include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol, and isopentanol.

In general, since the ion exchange group is introduced into the hydroxyl group on the surface of the separation material, the wet particles are drained by filtration or the like, immersed in an alkaline aqueous solution of a predetermined concentration, left for a certain time, and then water-organic. The halogenated alkyl compound is added and reacted in a solvent mixture system. This reaction is preferably carried out at a temperature of 40 to 90 ° C. for 0.5 to 12 hours. The ion exchange group to be provided is determined depending on the kind of the halogenated alkyl compound used in the above reaction.

As a method for introducing an amino group which is a weakly basic group as an ion exchange group, among the above halogenated alkyl compounds, a mono- having at least one alkyl group in which a part of hydrogen atoms is substituted with a chlorine atom. , Di- or tri-alkylamine, mono-, di- or tri-alkanolamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine, etc. A method is mentioned. The amount of these halogenated alkyl compounds used is preferably 0.2% by mass or more based on the total mass of the separating material. The reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.

As a method for introducing a strongly basic quaternary ammonium group as an ion exchange group, first, a tertiary amino group is introduced, and then the tertiary amino group is reacted with a halogenated alkyl compound such as epichlorohydrin. The method of converting into an ammonium group is mentioned. Further, quaternary ammonium hydrochloride or the like may be reacted with the separation material.

Examples of the method for introducing a carboxy group that is a weakly acidic group as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the halogenated alkyl compound. . The amount of the halogenated alkyl compound used is preferably 0.2% by mass or more based on the total mass of the separating material into which the ion exchange group is introduced.

As a method for introducing a sulfonic acid group which is a strongly acidic group as an ion exchange group, a glycidyl compound such as epichlorohydrin is reacted with a separating material, and a sulfite or a bisulfite such as sodium sulfite or sodium bisulfite. And a method of adding a separating material to the saturated aqueous solution. The reaction conditions are preferably 30 to 90 ° C. and 1 to 10 hours.

On the other hand, examples of the method for introducing ion exchange groups include a method in which 1,3-propane sultone is reacted with a separation material in an alkaline atmosphere. 1,3-propane sultone is preferably used in an amount of 0.4% by mass or more based on the total mass of the separating material. The reaction conditions are preferably 0 to 90 ° C. and 0.5 to 12 hours.

The moisture absorption of the separation material of this embodiment is measured by the following method. After 1 g of the dried separation material is left in a constant temperature and humidity test tank (temperature 60 ° C., humidity 90%) for 18 hours, the mass of the separation material is measured again to calculate the moisture absorption from the following equation.
(Separation material mass after moisture absorption -1) g / 1g x 100 = moisture absorption (%)

The moisture absorption of the separation material of the present embodiment is preferably 1 to 30% by mass, more preferably 1 to 20% by mass, and further preferably 1 to 10% by mass. When the moisture absorption of the separating material is 30% by mass or less, it is possible to suppress a decrease in liquid permeability of the separating material due to the thickness of the coating layer.

The average pore diameter, mode diameter, specific surface area and porosity in the pore diameter distribution of the separating material or porous polymer particles of the present embodiment are values measured with a mercury intrusion measuring apparatus (Autopore: manufactured by Shimadzu Corporation). Measured as follows. 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume: 0.4 mL) and measured under conditions of an initial pressure of 21 kPa (approximately 3 psia, corresponding to a pore diameter of approximately 60 μm). Mercury parameters are set to a device default mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes / cm. Each value is calculated by limiting the pore diameter to a range of 0.1 to 3 μm.

The separation material of this embodiment is suitable for use in separation of proteins by electrostatic interaction and affinity purification. For example, after adding the separation material of the present embodiment to a mixed solution containing protein, adsorbing only the protein to the separation material by electrostatic interaction, the separation material is filtered from the solution, and the salt concentration If added to a high aqueous solution, the protein adsorbed on the separation material can be easily desorbed and recovered. Moreover, the separation material of this embodiment can also be used in column chromatography. FIG. 1 shows an embodiment of a separation column. The separation column 10 includes a column 1 and a separation material 2 packed in the column 1.

As the biopolymer that can be separated using the separation material of the present embodiment, a water-soluble substance is preferable. Specific examples include proteins such as blood proteins such as serum albumin and immunoglobulin, enzymes present in the living body, protein bioactive substances produced by biotechnology, DNA, biopolymers such as bioactive peptides, etc. Yes, preferably the weight average molecular weight is 2 million or less, more preferably 500,000 or less. In addition, according to a known method, it is necessary to select properties, conditions, etc. of the separation material depending on the isoelectric point, ionization state, etc. of the protein. Examples of known methods include the methods described in JP-A-60-169427.

In the separation material of the present embodiment, after the coating layer on the porous polymer particles is cross-linked, an ion exchange group, protein A, etc. are introduced into the surface of the separation material, thereby separating natural macromolecules such as proteins. Each has the advantage of particles made of a polymer or particles made of a synthetic polymer. In particular, since the porous polymer particles in the separation material of the present embodiment are obtained by the above-described method, they have durability and alkali resistance. In addition, the separation material of the present embodiment tends to reduce non-specific adsorption of proteins and easily cause protein adsorption / desorption. Furthermore, the separation material of the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of protein or the like under the same flow rate.

In the present specification, the liquid flow rate represents the liquid flow rate when the separation material of this embodiment is filled in a stainless steel column of φ7.8 × 300 mm and the liquid is passed. When the separation material of this embodiment is packed in a column, it is preferable that the liquid passing speed is 800 cm / h or more when the column pressure is 0.3 MPa. When separating proteins by column chromatography, the flow rate of protein solution or the like is generally in the range of 400 cm / h or less. However, when the separation material of the present embodiment is used, the separation rate for normal protein separation is as follows. It can be used at a liquid passing speed of 800 cm / h or more faster than the separating material.

The average particle size of the separation material of this embodiment is preferably 10 to 300 μm. For use in preparative or industrial chromatography, it is preferably 10 to 100 μm in order to avoid an extreme increase in column internal pressure.

When the separation material of this embodiment is used as a column packing material in column chromatography, it has excellent operability because there is almost no volume change in the column regardless of the properties of the eluate used.

The 5% compressive deformation elastic modulus of the separating material of the present embodiment can be calculated as follows.
Using a micro compression tester (Fisher), the load when particles are compressed to 50 mN with a smooth end face (50 μm × 50 μm) of a quadrangular prism at room temperature (25 ° C.) at a load rate of 1 mN / sec. And measure the compression displacement. From the measured values obtained, the compression modulus (5% K value) when the particles are compressively deformed by 5% can be obtained by the following formula. Further, the load at the point at which the displacement during the measurement changes most greatly is defined as the breaking strength (mN).
5% K value (MPa) = (3/2 ^1/2 ) · F · S ⁻³ / ² · R ^−1/2
F: Load (mN) when the crosslinked polymer particles are 40% compressively deformed
S: Compression displacement (mm) when the crosslinked polymer particles are 40% compressed and deformed
R: radius of cross-linked polymer particles (mm)

The compression elastic modulus (5% K value) when the separating material is 5% compressively deformed is preferably 100 to 1000 MPa, more preferably 200 to 1000 MPa, and further preferably 250 to 1000 MPa.

The pore volume (porosity) of the separation material is preferably 30% by volume or more and 70% by volume or less, based on the total volume (including the pore volume) of the separation material, and is 40% by volume or more and 70% by volume or less. It is more preferable. The separating material preferably has pores having an average pore diameter of 0.05 to 1 μm. The average pore diameter is preferably 0.2 to 0.5 μm. If the pore diameter is 0.05 μm or more, a substance tends to easily enter the pores. If the pore diameter is 1 μm or less, the specific surface area is sufficient.

The specific surface area of the separating material is preferably 10 m ² / g or more, and more preferably 30 m ² / g or more. From the viewpoint of higher practicality, the specific surface area is more preferably 35 m ² / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 10 m ² / g or more, the adsorption amount of the substance to be separated tends to increase.

The 5% deformation modulus, mode diameter in the pore size distribution, specific surface area, etc. of the separating material can be adjusted by appropriately selecting the raw material of the porous polymer particles, the porosifying agent, the polymer having a hydroxyl group, and the like. it can.

In addition, although this embodiment demonstrated the separation material of the form which introduce | transduces an ion exchange group, even if it does not introduce | transduce an ion exchange group, it can use as a separation material. Such a separating material can be used for, for example, gel filtration chromatography. That is, the separation column of this embodiment includes a column and the separation material of this embodiment packed in the column.

Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

(Example 1)
<Synthesis of porous polymer particles>
To a 500 mL three-necked flask, add 16 g of 96% pure divinylbenzene (manufactured by Nippon Steel & Sumikin Chemical Co., Ltd., trade name: DVB960), 19.5 g of isoamyl alcohol, 4.5 g of diethylbenzene, and 0.64 g of benzoyl peroxide. An aqueous solution of tricalcium phosphate (TCP) (0.5% by mass) was prepared. This aqueous solution was emulsified using a microprocess server, and the resulting emulsion was transferred to a flask and stirred for about 8 hours using a stirrer while heating in a water bath at 80 ° C. The obtained particles were filtered and washed with acetone. Finally, TCP was dissolved with an acidic aqueous solution to obtain porous polymer particles 1. The specific surface area of the obtained particles and the mode diameter in the pore size distribution were measured by a mercury intrusion method, and the average particle size was measured by a flow type particle size measuring device. The results are shown in Table 1.

<Formation and cross-linking of coating layer>
4 g of sodium hydroxide and 0.14 g of glycidyl phenyl ether were added to 100 mL of an agarose aqueous solution (2% by mass) and reacted at 70 ° C. for 12 hours to introduce a phenyl group into agarose. The obtained modified agarose was reprecipitated with isopropyl alcohol and washed.
Porous polymer particles 1 are added to a 200 mg / mL modified agarose aqueous solution, and 1 g of porous polymer particles 1 are added to 70 mL of the aqueous solution, and the mixture is stirred at 50 ° C. for 24 hours. Adsorbed. After adsorption, it was filtered and washed with hot water for 8 hours.
The results are shown in Table 1.
The modified agarose was crosslinked as follows. 1 g of porous polymer particles 1 on which modified agarose is adsorbed with respect to 35 mL of the aqueous solution are added to an aqueous solution having ethylene glycol diglycidyl ether and sodium hydroxide concentrations of 0.64 M and 0.4 M, respectively, for 24 hours. Stir at room temperature. Then, after washing | cleaning with the heated 2 mass% sodium dodecyl sulfate aqueous solution, it wash | cleaned with the pure water and dried, and the separating material was obtained.

(Measurement of coating amount on porous polymer particles with modified agarose)
The amount of agarose adsorbed on the porous polymer particles was measured by weight reduction due to thermal decomposition. 10 mg each of porous polymer particles, modified agarose, and particles (separation material) obtained by the formation and crosslinking of the coating layer were heated from 30 ° C to 900 ° C. Since it was found that the porous polymer particles were thermally decomposed at 500 ° C. and the modified agarose was thermally decomposed at 300 ° C., the modified agarose coating amount of the modified agarose-coated porous polymer particles was estimated from these two data.
Specifically, the weight loss of the modified agarose and the porous polymer particles was measured by thermogravimetric analysis, and the amount of the modified agarose per 1 g of the porous polymer particles was calculated. The coating amount per unit specific surface area was calculated by dividing the amount of modified agarose per 1 g of the porous polymer particles calculated here by the specific surface area (see the following formula (1)). The results are shown in Table 1.
Coating amount per unit specific surface area [mg / m ² ] = (weight reduction amount of polymer having hydroxyl group [%]) / (weight reduction amount of porous polymer particle [%]) / (specific surface area [m ² / g]) × 1000 (1)

(Evaluation of nonspecific adsorption ability of protein)
First, a calibration curve was prepared with a known concentration of BSA (bovine serum albumin) aqueous solution (0 to 0.4%). 200 mg of particles (separation material) obtained by the formation and crosslinking of the coating layer were immersed in water at 20 ° C. for 12 hours to swell. The swollen particles and 10 mL of water were weighed into a vial. BSA was weighed into a 100 mL volumetric flask and dissolved in a phosphate buffer solution (pH = 7.1) to prepare 12 mg / mL and 24 mg / mL BSA aqueous solutions. 10 mL of these BSA solutions were added to the particle solution and stirred with a mix rotor at 25 ° C. for 24 hours. 10 mL of the supernatant of this solution was diluted 10 times, the absorbance (280 nm) was measured, and the amount of BSA adsorbed on the separation material was calculated as the amount of non-specific adsorption. The results are shown in Table 2.

(Measurement of oxygen element ratio)
The particles (separation material) obtained by the formation and crosslinking of the coating layer were dried with a dryer at 70 ° C. for 12 hours, and then the oxygen element ratio on the particle surface was measured with the following XPS (X-ray photoelectron spectroscopy) apparatus. . The results are shown in Table 1.
Moreover, after drying particle | grains by the same method, particle | grains were ground with the mortar and the oxygen element ratio inside particle | grains was measured with the following XPS apparatus. The results are shown in Table 1.
The oxygen element ratio was calculated by dividing the peak intensity derived from the oxygen element by the peak intensity of an element other than the hydrogen element.
XPS device: PHI 5000 VersaProbeII manufactured by ULVAC-PHI
X-ray: Monochromatic AIkα ray (1486.6 eV))
Analysis area: 200 μm ²
Analysis depth: 50 mm

<Introduction of ion exchange groups>
The particles (separation material) obtained by the formation and crosslinking of the coating layer were subjected to centrifugation to remove water, and then poured into a mixture of 5 M sodium hydroxide aqueous solution 20 mL and 5 M diethylaminoethyl hydrochloride aqueous solution 20 mL, The mixture was stirred at 70 ° C. for 12 hours. After completion of the reaction, the mixture was filtered and washed with water to obtain a DEAE-modified separation material having a diethylaminoethyl (DEAE) group as an ion exchange group.

(Evaluation of protein binding capacity and recovery rate)
200 mg of the obtained DEAE-modified separating material was immersed in water at 20 ° C. for 12 hours to swell. The swelling separator and 10 mL of water were weighed into a vial. Bovine serum albumin (BSA) was weighed into a 100 mL volumetric flask and dissolved in a phosphate buffer solution (pH = 7.1) to prepare 1.2 mg / mL and 12 mg / mL BSA aqueous solutions. 10 mL of this BSA solution was added to the vial that weighed the swelling separator and stirred with a mix rotor at 25 ° C. for 24 hours. 10 mL of the supernatant of this solution was diluted 10-fold, the absorbance (280 nm) was measured, and the BSA adsorption amount (binding capacity) to the DEAE-modified separation material was calculated. The results are shown in Table 2.

NaCl 0.9g was added to this vial and stirred at 25 ° C for 12 hours with a mix rotor. The supernatant (5 mL) of this solution was diluted 10-fold, the absorbance (280 nm) was measured, and the BSA recovery rate by the separating material was calculated as shown in Equation (2). The results are shown in Table 2.
Protein recovery rate = (absorbance after protein desorption−absorbance before protein desorption) / (absorbance after protein adsorption−absorbance before protein adsorption) (2)

(Quantification method of ion exchange groups)
DEAE-modified separation material 0.2 to 0.3 g swollen with water for 12 hours or more was quantified, transferred to a beaker, and 20 mL of 0.1 N sodium hydroxide solution was added and stirred. Thereafter, suction filtration was performed, and the particles on the filter were washed until the washing solution became neutral. Then, it moved to the beaker, 20 mL of 0.1N hydrochloric acid aqueous solution was added, and it stirred at room temperature for 1 hour. Thereafter, suction filtration was performed, and the particles on the filter were washed until the washing solution became neutral. This washing solution was titrated with a 0.1N aqueous sodium hydroxide solution using an automatic potentiometric titrator, and the ion exchange capacity was measured. The results are shown in Table 2.

(Column characteristic evaluation)
The obtained DEAE-modified separation material was packed in a stainless steel column of φ7.8 × 300 mm as a slurry (solvent: methanol) having a concentration of 30% by mass over 15 minutes. Thereafter, water was allowed to flow through the column while changing the flow rate, the relationship between the linear flow rate and the column pressure was measured, and the linear flow rate (liquid flow rate) at 0.3 MPa was measured. The results are shown in Table 1.

(Example 2)
<Coating layer formation and crosslinking> Synthesis and evaluation were performed in the same manner as in Example 1 except that epichlorohydrin was used instead of ethylene glycol diglycidyl ether.

(Example 3)
<Synthesis of porous polymer particles> Synthesis and evaluation were performed in the same manner as in Example 2 except that the amounts of isoamyl alcohol and diethylbenzene were changed to 16 g and 8 g, respectively.

(Comparative Example 1)
The same evaluation as in Example 1 was performed using the porous polymer particles 1 as they were.

(Comparative Example 2)
<Coating layer formation and cross-linking> Synthesis and evaluation were performed in the same manner as in Example 1 except that the concentration of the modified agarose aqueous solution was changed to 20 mg / mL.

(Comparative Example 3)
Commercially available agarose particles (Capto DEAE: GE Healthcare) were used as Comparative Example 3, and the same evaluation as in Example 1 was performed.

As is apparent from Table 1, when the linear flow rates were compared between the case where the synthetic polymer particles of Examples 1, 2 and 3 and Comparative Examples 1 and 2 were used, and the case where the agarose particles of Comparative Example 3 were used, the synthesis The polymer particles were larger and the liquid permeability was better. However, unless coating with a polymer having a hydroxyl group was performed, a large amount of non-specific adsorption occurred as in Comparative Example 1, and the protein was denatured. The comparative example 2 reduces the coating amount by the polymer which has a hydroxyl group. In this case as well, as in Comparative Example 1, a large amount of non-specific adsorption occurred and the protein was denatured. In Example 1, the coating amount with the polymer having a hydroxyl group is set within the predetermined range of the present invention. As a result, non-specific adsorption and protein denaturation can be suppressed, and the protein binding capacity and the protein recovery rate were improved as compared with those using agarose particles. Further, Example 2 is different from Example 1 in that the crosslinking agent is changed. In Example 2, nonspecific adsorption and protein denaturation were suppressed as in Example 1, and the ion exchange capacity was further increased and the protein binding capacity was improved. And Example 3 increases the coating amount per unit specific surface area of particle | grains rather than Example 2. FIG. In Example 3, as in Example 2, non-specific adsorption and protein denaturation were suppressed, the ion exchange capacity was further increased, and the protein binding capacity and the protein recovery rate were greatly improved.

1 ... column, 2 ... separation material, 10 ... column for separation.

Claims

Porous polymer particles containing at least one of styrene and divinylbenzene as a monomer unit in an amount of 90% by mass or more based on the total amount of monomers;
A coating layer containing a polymer having a hydroxyl group, covering at least a part of the surface of the porous polymer particles,
A separation material in which the coating amount by the coating layer is 1 to 15 mg / m 2 per unit specific surface area of the porous polymer particles.
The separation material according to claim 1, wherein the oxygen element ratio on the surface of the separation material is 10 to 50%.
The separation material according to claim 1 or 2, wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
The separation material according to claim 3, wherein the polysaccharide is at least one selected from agarose, chitosan, alginic acid, and dextran.
The separation material according to any one of claims 1 to 4, wherein the polymer having a hydroxyl group is crosslinked.
The separation material according to any one of claims 1 to 5, wherein a specific surface area of the porous polymer particles is 10 m 2 / g or more.
The separation material according to any one of claims 1 to 6, wherein a mode diameter in a pore size distribution of the porous polymer particles is 0.05 to 1 µm.
A separation column comprising a column and the separation material according to any one of claims 1 to 7 packed in the column.